Menaquinone-9 nanoparticles and treatment method

Abstract

This application discloses compositions comprising vitamin K2 nanoparticles and methods of using the same. In one embodiment, a composition is provided comprising water-soluble vitamin K2 nanoparticles (or nano-sized particles), wherein the nanoparticles have an average particle size of about 0.1 nm to 1,000 nm. In one embodiment, the vitamin K2 is MK-9 (menaquinone-9) and / or MKH2-9 (menaquinol-9). In another embodiment, the nanoparticles have an average particle size of 200 nm, 175 nm, 150 nm, 125 nm, 115 nm, 100 nm, 90 nm, less than 80 nm, or less than 75 nm. In another embodiment, the composition is a stable composition.

Description

Technical Field

[0001] Related Applications This application is related to U.S. Provisional Application No. 63 / 447,312, filed on February 21, 2023.

[0002] Field of the Invention The present invention relates to nanoparticles of menaquinone-9, compositions and formulations thereof for treating diseases related to vitamin K.

Background Art

[0003] Background of the Invention Vitamin K is known as a group of structurally similar fat-soluble vitamins. Vitamin K2 (or menaquinone) or menaquinone can be subdivided into nine related compounds, including short-chain menaquinones (such as menaquinone-4 or MK-4) and long-chain menaquinones (such as MK-7, MK-8, MK-9 to 14). Vitamins include phylloquinone (K1), menaquinone (K2) and menadione (K3). While plants synthesize vitamin K1, bacteria can produce a range of vitamin K2 forms, including the conversion of K1 to K2 by intestinal bacteria. Vitamin K3 is a synthetic version of the vitamin and is prohibited for human use by the U.S. Food and Drug Administration due to its toxicity.

[0004] It has been established that taking broad-spectrum antibiotics can reduce vitamin K production in the intestine, in humans, by almost 74% compared to humans who are not taking these antibiotics. A diet low in vitamin K also reduces the body's vitamin K concentration. Vitamin K1 is preferentially used by the liver as a coagulation factor. Vitamin K2 is preferentially used in the brain, vasculature, breast and kidneys. Vitamin K2 contributes to the production of myelin and sphingolipids (fats essential for brain health) and protects against oxidative damage in the brain. Vitamin K2, such as MK-4, promotes bone health by stimulating the production of connective tissue in bone. <0​ In animals, vitamin K2, the primary storage form, has several subtypes, which differ in the length of the isoprenoid group or the residues in the side chain. These vitamin K2 homologs are called menaquinones and are characterized by the number of isoprenoid residues in their side chains. For example, MK-4 has four isoprene residues in its side chain and is the most common type of vitamin K2 in animal products. MK-4 is usually synthesized from vitamin K1 in certain animal tissues (arterial walls, pancreas, and testes) by replacing the phytyl group with an unsaturated geranyl group containing four isoprene units. Unlike MK-4, MK-7 is not produced by human tissue. MK-7 can be converted from phylloquinone (K1) in the colon by E. coli bacteria. MK-4 and MK-7 are marketed in the United States as dietary supplements for bone health. MK-4 has been shown to reduce the incidence of fractures. In Japan, since 1995, MK-4 at a daily dose of 45 mg has been approved by the Ministry of Health for the prevention and treatment of osteoporosis.

[0006] Cardiovascular disease (CVD) is established as the leading cause of death in patients with chronic kidney disease (CKD). Compared to the general population, the risk of death due to CVD is approximately 10 to 20 times higher in CKD patients who are undergoing hemodialysis. Furthermore, vascular calcification and associated arteriosclerosis have been demonstrated to be frequently associated with the development of CVD. Therefore, the methods of treatment disclosure can be applied to the treatment of peripheral artery disease. In addition, CKD patients undergoing dialysis have a three times higher risk of fractures, including vertebral fractures and other types of fractures.

[0007] Vitamin K, including menaquinone-9 or MK-9, is present in low concentrations in a normal diet. A direct correlation between the level of vitamin K in a patient's blood and the incidence of vascular calcification, bone mineral density, and bone strength has also been established. Therefore, the adjunctive use of vitamin K, such as MK-9 and / or MKH2-9 (MK-9 / MKH2-9), as a nanoparticle formulation as disclosed herein, can increase bone mineral density or raise bone mineral density, in part, providing significant clinical benefits in reducing vascular calcification observed depending on the degree of arteriosclerosis, which can be helpful in the treatment or prevention of CVD and in the treatment or prevention of bone disease in patients with CKD. In one embodiment, a disclosed method for administering MK-9 as a nanoparticle can be used in the treatment or reduction of vascular calcification, in raising bone mineral density, and in the treatment, mitigation, or prevention of bone disease in patients with CKD, among others.

[0008] In food, vitamin K1 is established to be bound to the chloroplast membrane of green leafy vegetables. MK-4, derived from the conversion of menadione, a synthetic analog of vitamin K, is found in animal products such as eggs and meat. Long-chain menaquinones such as MK-7, MK-8, and MK-9 are found in fermented foods such as cheese, curd, and sauerkraut. The effect of long-chain MK-n, such as MK-7, on normal blood clotting is also established to be greater and longer-lasting than that of vitamin K1 and MK-4. MK-7 has also been shown to have a longer half-life in serum compared to MK-4 and yields superior carboxylation grade osteocalcin compared to vitamin K1. See Sato et al., Nutrition Journal, 2012, 11:93.

[0009] In humans, it can be established that nutritional doses of MK-9 are well absorbed, resulting in a significant increase in serum levels of MK-9. [ka]

[0010] It has been found that more than one-third of the drugs listed in the United States Pharmacopeia are either water-insoluble or poorly water-soluble. Furthermore, more than 40% of drugs are insoluble in the human body, which is significant considering that there are more than 5,000 small molecules under development. Solubility and stability are two of the most challenging properties that hinder drug development. In addition, water solubility is required to formulate many organic compounds under development as pharmaceuticals. Conventional formulation systems for highly insoluble drugs have required the application of combinations of organic solvents, surfactants, and emulsions, among other methods. For example, poorly water-soluble drugs such as vitamin K2 or MK-9 are usually excreted from the gastrointestinal tract before they can be absorbed into the bloodstream. It is known that the dissolution rate of certain compounds or drugs can increase with increasing surface area or decreasing particle size. Therefore, there has been a great deal of focus on the development of nanoparticles for delivering insoluble drugs, drugs with low solubility, or drugs with poor solubility. Nanoparticles are generally considered to be solid particles with a diameter of approximately 1 nm to 1000 nm. The above-mentioned examples of related technology and limitations are illustrative and not intended to be prescriptive. Other limitations of the related technology will be obvious to those skilled in the art upon reading this specification and examining the drawings or figures presented herein. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Sato et al., Nutrition Journal, 2012, 11:93 [Overview of the project] [Means for solving the problem]

[0012] Summary of the Invention Therefore, there is a continuing need for formulations effective for these indications. The following embodiments, aspects thereof, and variations are illustrative and not intended to limit the scope.

[0013] In one embodiment, a composition is provided comprising water-soluble vitamin K2 nanoparticles (or nano-sized particles), wherein the nanoparticles have an average particle size of about 0.1 nm to 1,000 nm. In one embodiment, the vitamin K2 is MK-9 (menaquinone-9) and / or MKH2-9 (menaquinol-9). In another embodiment, the nanoparticles have an average particle size of 200 nm, 175 nm, 150 nm, 125 nm, 115 nm, 100 nm, 90 nm, less than 80 nm, or less than 75 nm. In another embodiment, the composition is a stable composition.

[0014] In one variant, the nanoparticles have an average particle size range of approximately 10nm-750nm, 20nm-700nm, 40nm-600nm, 50nm-500nm, 60nm-400nm; or approximately 45nm-95nm. In another variant, the nanoparticles have an average particle size range of approximately 70nm-300nm, 80nm-200nm, 90nm-175nm, 100nm-150nm, or approximately 120nm-130nm. In yet another variant, the nanoparticles have an average particle size range of approximately 75nm-175nm, 85nm-165nm, 95nm-155nm, 105nm-145nm, 145nm-175nm, or approximately 155nm-165nm. In yet another variant, the nanoparticles have an average particle size range of approximately 75nm-105nm and 85nm-90nm. In another variant, the average particle size is approximately 155 nm.

[0015] In another embodiment of the above composition, the nanoparticles are prepared using a homogenizer selected from the group consisting of a rotor stator homogenizer, a bead mill homogenizer, or a mortar and pestle homogenizer. In one variant, the nanoparticles are prepared using a milling process such as wet milling, wet milling using a high-pressure homogenizer, a dry milling process, or jet milling. See T. Niwa et al., Universal wet-milling technique to prepare oral nanosuspension focused on discovery and preclinical animal studies - Development of particle design method, International Journal of Pharmaceutics, Vol. 405, 1-2, 28 February, 2011, 218-227; T. Niwa et al., Design of Dry Nanosuspension with Highly spontaneous Dispersible Characteristics to Develop Solubilized Formulation for Poorly water-Soluble Drugs, Pharmaceutical Research, 28, 2339-2349, 2011.

[0016] In another embodiment, the composition contains poloxamer 188, polysorbate 80, polysorbate 20, Vit E-TPGS (TPGS), TPGS-1000, TPGS-750-M, Solutol HS15, PEG-40 hydrogenated castor oil, PEG-35 castor oil, PEG-8 caprylic / capric glyceride, PEG-32 glyceryl laurate, PEG-32 glyceryl palmitostearate, polysorbate 85, polyglyceryl-6 dioleate, sorbitan monooleate, and Capmul. The present invention further comprises at least one emulsifier selected from the group consisting of MCM, Maisine 35-1, glyceryl monooleate, glyceryl monolinoleate, PEG-6 oleate, PEG-6 linoleate, oleic acid, linoleic acid, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, polyglyceryl-3 diisostearate, and lecithin. In another embodiment, the emulsifier is selected from polysorbate 80, Vit E-TPGS, Solutol HS15, PEG-40 hydrogenated castor oil, and PEG-35 castor oil.

[0017] In one embodiment, the composition further comprises at least one bioavailability enhancer selected from the group consisting of medium-chain fatty acids, omega-3 fatty acids, capric acid, caprylic acid, alkyl glycosides, chitosan, trimethylated chitosan, ethylene glycoltetraacetic acid, ethylenediaminetetraacetic acid, salicylic acid, genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one) and pharmaceutically acceptable salts thereof.

[0018] In another embodiment, the composition is a nanosuspension in water. In yet another embodiment, the composition or nanosuspension is at least five times more soluble than commercially available (chemically synthesized or fermented) unhomogenized vitamin K2 or vitamin K-2 that is not formed or prepared as nanoparticles such as MK-9 and / or MKH2-9 (menaquinol-9). In one variant, the composition is at least two, three, five, seven, ten, fifteen, twenty times or more soluble than commercially available unhomogenized vitamin K2. As used herein, “unhomogenized” vitamin K2 means commercially available vitamin K2 such as MK-9 that is not homogenized, not milled, or otherwise prepared as nanoparticles or nanosuspensions as described herein. In another embodiment, vitamin K2 is MK-9. In another embodiment, the vitamin K2 nanosuspension is prepared or carried out at a concentration of 0.01 mg / mL in water. In yet another embodiment, the nanosuspends are present in an aqueous solution of Fed State Simulated Intestinal Fluid (FeSSIF). In yet another embodiment, the solubility is determined after 10 minutes in FeSSIF.

[0019] In another embodiment, the composition further comprises pharmaceutically acceptable additives, and the composition is effective for the treatment of vitamin K-related conditions, or for the treatment of osteoporosis or arteriosclerosis.

[0020] In another embodiment, there is provided a method for treating a disease selected from the group consisting of neurodegenerative diseases, retinopathies, rheumatoid polyarthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, aging of the skin and pathologies associated with aging in tissues, pathologies associated with mitochondrial dysfunction, cachexia associated with malnutrition in a mammal, the treatment being accompanied by an increase in the lifespan of the mammal, the method comprising administering a therapeutically effective amount of any of the above compositions. Accordingly, there is provided a compound or composition disclosed herein for use in treating a disease selected from the group consisting of neurodegenerative diseases, retinopathies, rheumatoid polyarthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, aging of the skin and pathologies associated with aging in tissues, pathologies associated with mitochondrial dysfunction, cachexia associated with malnutrition in a mammal, the treatment being accompanied by an increase in the lifespan of the mammal.

[0021] In another embodiment, a method for treating a mammal is provided, which includes administering to a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative diseases, and cardiovascular diseases, any of the above compositions in a therapeutically effective amount. In yet another embodiment, a method for treating or preventing osteoporosis and / or osteopenia is provided, which includes administering to a patient in need of treatment a therapeutically effective amount of the above composition. In yet another embodiment, a method for treating, preventing, slowing the progression of, arresting, and / or reversing calciphylaxis in a mammal that requires treating, preventing, slowing the progression of, arresting, and / or reversing calciphylaxis is provided, which includes administering to the mammal any of the above compositions in a therapeutically effective amount and a pharmaceutically acceptable additive to prevent, slow the progression of, arrest, or reverse calciphylaxis. In one aspect, the mammal has distal calciphylaxis and / or central calciphylaxis. In another aspect, the mammal has diabetes, chronic kidney disease, or end-stage kidney disease.

[0022] In one variant of the above method, the mammal has stage 3, stage 4, or stage 5 chronic kidney disease. In another variant of the method, the mammal is undergoing hemodialysis. In another variant of the method, the mammal is receiving non-warfarin anticoagulant therapy. In another variant of the method, the anticoagulant therapy is oral anticoagulant therapy. In yet another variant of the method, the anticoagulant therapy includes an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eliquis, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0023] Treatment of COPD: Chronic obstructive pulmonary disease (COPD) is a term used to describe a progressive lung disease that makes breathing difficult. The two main forms of COPD are emphysema and chronic bronchitis. Furthermore, elastinolysis (the breakdown of elastin proteins) is a key feature of COPD. COPD contributes to the loss of arterial flexibility and promotes calcification of the intimal media of blood vessels. It has also been shown to be a strong predictor of mortality in patients with COPD (Rabinovich et al., (2016) Circulating desmosine levels do not predict emphysema progression but are associated with cardiovascular risk and mortality in COPD, ERJ Express doi: 10.1183 / 13993003.01824-2015). MGP has been shown to inhibit the production of matrix metalloproteinases that promote elastinolysis. Vitamin D may be a key determinant of the rate of elastin degradation, and low vitamin D levels result in low MGP activity, which is inadequate for protection against elastofibrosis (Piscaer et al., (2017) Vitamin D deficiency: the linking pin between COPD and cardiovascular diseases? RESP. RES. 18:189). While we do not wish to be constrained by theory, the administration of vitamin K2 disclosed herein may increase the production of activated (carboxylated) MGP, thereby suppressing the adverse effects of elastofibrosis in subjects with COPD, thereby preventing, slowing, or reversing the progression of one or more symptoms of COPD. Furthermore, treatment of elastin degradation may be effective in treating Covid-19 and its variants. Therefore, the nanoparticle formulations disclosed herein may be administered to treat or prevent elastin degradation and diseases associated with elastin degradation.

[0024] In any one aspect of the above method, the mammal has chronic obstructive pulmonary disease (COPD). In another aspect of the method, the mammal has calciphylaxis-related skin lesions. In one variant of the method, administration of the composition reduces the total surface area of ​​the skin lesions by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In another variant of the method, administration of the above composition to the mammal increases the mammal's serum T50 level by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the mammal's serum T50 level before administration of the composition. In another variant of the method, administration of the composition increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the mammal's plasma after administration of the composition, to a higher level than before administration of the composition.

[0025] In one embodiment, a method is provided for treating, preventing, slowing, stopping, and / or reversing the progression of tissue calcification or calciphylaxis in a mammal (or subject), the method comprising the step of administering at least 0.1 mg of the composition per day to the mammal to prevent, slow the progression of, and / or stop tissue calcification, including soft tissue calcification, wherein the composition is administered in the form of a pharmaceutical composition. In another embodiment, a method is provided for treating, preventing, slowing, stopping, and / or reversing tissue calcification in a prediabetic mammal (or subject) having diabetes mellitus, chronic kidney disease, or a combination thereof, which requires treatment, prevention, slowing, cessation, and / or reversal of its progression, wherein the method comprises the step of administering to the mammal at least 0.01 mg or at least 0.1 mg per day of any one of the above compositions, wherein the above compositions are administered in a pharmaceutical composition. In one variant of the method, the mammal has diabetes mellitus. In another variant, the mammal has type II diabetes mellitus. In another variant, the mammal has been diagnosed with prediabetes. In one embodiment, the mammal has chronic kidney disease. In one variant of the above method, the mammal has stage 4 or stage 5 chronic kidney disease / end-stage renal disease. In another variant of the method, the mammal is undergoing hemodialysis. In another variant, mammals receive anticoagulant therapy that is not warfarin-based. In another variant, the anticoagulant therapy is oral anticoagulant therapy. In yet another variant, the anticoagulant therapy includes an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0026] In another variant, a method is provided for treating, preventing, slowing, stopping, and / or reversing tissue calcification in a mammal undergoing hemodialysis and requiring treatment, prevention, slowing, stopping, and / or reversing of its progression, wherein the method comprises the step of administering at least 0.01 mg or at least 0.1 mg of the composition per day to the mammal, thereby preventing, slowing, stopping, and / or reversing its progression, the composition being administered in a pharmaceutical formulation. In one variant of the method, the mammal has diabetes. In another aspect, the application discloses a fortified food formulation or beverage formulation comprising adding a composition comprising any one of the compositions disclosed herein to a food or beverage.

[0027] In another embodiment of the present method, the proliferative disorder is selected from the group consisting of cancer, leukemia, and inflammatory diseases. In another embodiment, a method is provided for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative disorders, and cardiovascular diseases, comprising the step of administering to the mammal a therapeutically effective amount of any of the above compositions. In one embodiment, the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, leukemia, neuroblastoma, glioblastoma, cervical cancer, colorectal cancer, pancreatic cancer, bladder cancer, renal cancer, prostate cancer, ovarian cancer, and head and neck cancer.

[0028] In another embodiment, a method is provided for treating, preventing, slowing, stopping, and / or reversing the progression of Alzheimer's disease (AD) in a mammal or subject that requires treatment, prevention, slowing, stopping, and / or reversing the progression of the disease, the method comprising the step of administering to the mammal or subject at least 0.01 mg or at least 0.1 mg of any of the above compositions per day to prevent, slow the progression of, and / or stop, or reverse Alzheimer's disease.

[0029] In another aspect, the application discloses a pharmaceutical composition comprising a menaquinone composition disclosed in a therapeutically effective amount and a pharmaceutically acceptable additive, which is effective for the treatment of a vitamin K-related condition selected from the treatment of osteoporosis and arteriosclerosis.

[0030] In another aspect of the above method, the anticoagulant therapy is oral anticoagulation therapy. In another aspect, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban. In another aspect, the mammal has chronic obstructive pulmonary disease (COPD). In another aspect, the mammal has calciphylaxis-related skin lesions. In another aspect of the method, administration of the composition reduces the total surface area of ​​the skin lesions by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In another embodiment of this method, administration of the composition disclosed herein to a mammal results in an increase in the mammal's serum T50 level by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the mammal's serum T50 level before administration of the disclosed composition. In another embodiment, administration of the disclosed composition increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the mammal's plasma after administration of the composition, to a level higher than before administration of the composition. In one embodiment of this method, the increase in the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in mammalian plasma after administration of the composition is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the ratio before administration.

[0031] In certain embodiments described above, administration of the disclosed composition reduces the amount of non-carboxylated vitamin K-dependent proteins in the plasma of the subject by, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the amount before administration of the composition. In certain variant forms, the vitamin K-dependent proteins are selected from matrix Gla protein (MGP), growth arrest-specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, activated protein S, factor II, factor VII, factor IX, and factor X.

[0032] In certain variations of the above method, administration of the composition increases the plasma levels of osteoprotegerin or fetuin A by, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the plasma concentration of osteoprotegerin or fetuin A before administration of the composition. In other variant forms, administration of the composition reduces plasma levels of D-dimers or highly sensitive C-reactive peptide (hs-CRP) by, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the plasma concentration of D-dimers or highly sensitive C-reactive peptide (hs-CRP) before administration of the composition.

[0033] In certain variations of the above method, the method may include a step of administering to a subject approximately 0.01 mg or 0.1 mg to approximately 200 mg of the composition per day. In other variations, the method may include a step of administering to a subject approximately 0.1 mg to approximately 150 mg of the composition per day. In other variations, the method may include a step of administering to a subject approximately 0.1 mg to approximately 100 mg of the composition per day. In other variations, the method may include a step of administering to a subject approximately 2 mg to approximately 200 mg of the composition per day. In certain variations, the method may include a step of administering to a subject approximately 2 mg to approximately 250 mg of the composition per day. In other variations, the method may include a step of administering to a subject approximately 2 mg to approximately 250 mg of the composition per day. In other variations, the method may include a step of administering to a subject approximately 2 mg to approximately 100 mg of the composition per day. In other variations, the method may include a step of administering to a subject about 3 mg to about 100 mg of the composition per day. In other variations, the method may include a step of administering to a subject about 0.5 mg to about 75 mg of the composition per day, for example, a step of administering to a subject 0.01 mg, 0.1 mg, 1 mg, 2 mg, 3 mg, or 10 mg of the composition per day.

[0034] In certain variants, the composition is administered to the subject for at least two weeks, four weeks, six weeks, eight weeks, three months, six months, one year, or indefinitely, as needed. If the subject is undergoing hemodialysis, the composition may be administered to the subject for at least the duration of the hemodialysis.

[0035] In another variation of the method for treating calciphylaxis, in addition to measuring the change / reduction in lesion size before and after drug administration following administration of the disclosed composition, biopsies of the associated lesions can be taken using von Kossa staining to determine evidence of changes in PTH at the tissue level and calcium and phosphate deposition in the cutaneous arterioles.

[0036] As disclosed herein, the presence of uremic oxidative blockade can be detected, for example, by detecting an increase in F2 isoprostane, leading to increased plasma lipid peroxidation (Morrow et al. (1990) A series of prostaglandin F2-like compounds are produced in vivo by humans by a non-cyclooxygenase, free radical-catalyzed mechanism, PROC. NATL. ACAD. SCI. USA 87:9383-9387), an increase in isolevuglandin-plasma protein adducts (Salomon et al. (2000) Isolevuglandin-protein adducts in humans: Products of free radical induced lipid oxidation through the isoprostane pathway, BIOCHIM BIOPHYS ACTA 1485:225-235), and an increase in exhaled ethane (Handelman et al. (2000) J AM. SOC. NEPHROL. 11:271A); For example, increased oxidation of proteins and amino acids by detecting oxidation of tyrosine residues (Heinecke et al. (1999) Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: Sensitive and quantitative assays for oxidized amino acids in proteins and tissues, METHODS ENZYMOL. 300:124-144), oxidation of cysteine ​​or methionine residues in plasma proteins, lysine oxidation and threonine oxidation, thiol oxidation and carbonyl formation (Himmelfarb et al. (2000) Plasma protein thiol oxidation and carbonyl formation in chronic renal failure, KIDNEY INT.58:2571-2578); for example, reactive aldehyde formation by detection of glyoxal, methylglyoxal, acrolein, glycoaldehyde and parahydroxyphenylacetaldehyde (phenacetaldehyde) (Miyata et al. (1999) Alterations in nonenzymatic biochemistry in uremia: Origin and significance of 'carbonyl stress' in long-term uremic complications. KIDNEY INT. 55:389-399); for example, increased reactive carbonyl compounds by measuring hydrazine formation after reaction with 2,4-dinitrophenylhydrazine; decreased plasma glutathione levels and glutathione peroxidase function (Ceballos-Picot et al. (1996) Glutathione antioxidant system as a marker of oxidative stress in chronic renal failure, FREE RADIC. BIOL. MED. This is determined by measuring the increase in the ratio of oxidized thiols to reduced thiols (Hultberg et al. (1995) Reduced, free, and, total fractions of homocysteine ​​and other thiol compounds in plasma from patients with renal failure, NEPHRON 70:62-67; Himmelfarb et al. (2002) Plasma aminothiol oxidation in chronic renal failure, KIDNEY INT 61:705-716; Ward et al. Polymorphonuclear leukocyte oxidative burst is enhanced in patients with chronic renal insufficiency, J AM. SOC. NEPHROL. 5:1697-1702).

[0037] In another embodiment, a method is provided for treating, preventing, slowing, stopping, and / or reversing tissue calcification in a prediabetic mammal (or subject) having diabetes mellitus, chronic kidney disease, or a combination thereof, which requires treatment, prevention, slowing, cessation, and / or reversal of its progression, wherein the method comprises the step of administering at least 0.01 mg of the disclosed composition to the mammal to prevent, slow the progression of, and / or cessation of tissue calcification, wherein the composition is administered as a pharmaceutical composition. In another embodiment of the method, the mammal has diabetes mellitus. In yet another embodiment, the mammal has type II diabetes mellitus, or the mammal has been diagnosed as prediabetic. In another embodiment, the mammal has chronic kidney disease. In yet another embodiment of the method, the mammal has stage 4 or stage 5 chronic kidney disease / end-stage renal disease. In yet another embodiment, the mammal is undergoing hemodialysis. In another embodiment, the mammal is undergoing anticoagulant therapy that is not warfarin-based. In another embodiment, the anticoagulant therapy is oral anticoagulation therapy. In another embodiment of the present method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0038] In another embodiment, a method is provided for treating, preventing, slowing, stopping, and / or reversing tissue calcification in a mammal undergoing hemodialysis and requiring treatment, prevention, slowing, stopping, and / or reversing of its progression, the method comprising the step of administering to the mammal at least 0.01 mg or 0.1 mg per day of substantially pure of the composition disclosed herein, thereby preventing, slowing, stopping, and / or reversing the tissue calcification, wherein the disclosed composition is administered as a pharmaceutical composition. In another embodiment, the mammal has diabetes.

[0039] Vitamin K Metabolism: Development of Vascular and Soft Tissue Calcification After Failure to Regenerate Reduced Vitamin K: Vitamin K is an essential enzymatic cofactor required for the post-translational modification of vitamin K-dependent (VKD) proteins. Several VKD proteins exist, but many are clinically associated with ESRD patients. These include central coagulation factors such as factor II, factor VII, factor IX, and factor X, as well as intercellular matrix proteins including matrix GLA-1 and osteocalcin. Under normal conditions, vitamin K is reduced to vitamin K hydroquinone (KH2) by the enzyme NADPH oxidase. Only reduced vitamin K can function as a cofactor for gamma glutamate carboxylase (GGCX), which catalyzes the carboxylation of vitamin K-dependent proteins. Warfarin blocks the production of vitamin K hydroquinone by acting as a reductive sink. Enzymatic carboxylation of glutamic acid residues further oxidizes vitamin KH2 to 2-3 epoxide vitamin K (Figure 2). The final step in the vitamin K cycle requires enzymatic oxidation of the vitamin K2-3 epoxide back to its native structure. This step is catalyzed by vitamin K oxidative reductase (VKOR), a component of the vitamin K cycle, and is also blocked by the oxidative action of warfarin. The observation that warfarin blocks both the production of vitamin K hydroxyquinone (KH2) and the regeneration of vitamin K2 2-3 epoxide helps explain why calciphylaxis and other forms of dystrophic calcification occur more frequently among patients receiving warfarin treatment.

[0040] In one variant form, supplementation with the disclosed composition reduces the risk of vascular and soft tissue calcification by increasing the formation of primary calciprotein particles (CPPs) consisting of fetuin A and carboxylated matrix GLA-1 protein. Under normal physiological conditions, plasma calcium and phosphate concentrations are nearly supersaturated and therefore expected to precipitate as crystalline hydroxyapatite in blood vessels and soft tissues. The observation that this process does not occur suggests the existence of potent chemical and biological means to block pathological calcification. Recent studies have shown that circulating calcium phosphate crystals form primary calciprotein particles (CPPs) by complexing with two calcification-inhibiting proteins. These protein-inorganic complexes are primarily composed of fetuin A (a liver-derived protein shown to prevent vascular calcification). A smaller amount of the second protein is matrix Gla-1 protein, which also functions to prevent pathological calcification. Matrix Gla-1 is a vitamin K-dependent protein, and early studies by Price et al. and others have shown that the formation of fetuin-matrix Gla-1 inorganic nanoparticles (primary calciprotein CPPs) depends on the gamma-carboxylation of matrix Gla-1. Preclinical studies suggest that the calciprotein system functions as an alternative means of preventing pathological calcification when humoral defenses such as pyrophosphate, magnesium, and albumin are breached. The "absorption" of calcium-phosphate crystals by primary CCPs occurs in a coordinated and time-dependent process.

[0041] Time to 50% saturation of the primary CCP (T) 50 ) is an accurate and highly sensitive means of determining the ability of plasma to “precipitate” or “absorb” excess calcium phosphate crystals. 50 Patients who have had a long time have a reduced ability to absorb calcium phosphate crystals, while long T 50 Patients who have time are consistent with having high capacity. Recent clinical trials have shown that T 50The validity of the test was proven, and the low T 50 The time has been confirmed to be associated with an increased risk of myocardial infarction, heart failure, and all-cause mortality. Therefore, any clinical intervention that can increase the synthesis of circulating primary CCPs will improve the ability to prevent pathological calcification. It should be noted that this process is essential for primary CPP formation, as patients with CKD and ESRD show reduced levels of carboxylated matrix Gla-1 protein. Therefore, the disclosed composition and supplementation or administration of the composition in patients with CKD or ESRD will reduce the risk of pathological calcification and prevent the development of vascular and soft tissue calcification.

[0042] By supplementing or administering the disclosed composition, the production of carboxylated matrix Gla-1 and GAS-6 can be restored, thereby preventing or slowing the development of soft tissue and vascular calcification in skin tissue.

[0043] Vitamin K regeneration involves two key enzymes: vitamin K2-3 epoxide oxidative reductase (VKOR) and NAD(P)H:quinone oxidative reductase (NQO1). As shown in the figure, VKOR reduces 2-3 vitamin K epoxides to vitamin K quinone, while NADPH reduces vitamin K quinone to its hydroxyquinone form (KH2). Recent studies have shown that VKOR has two distinct isoforms (VKORC-1 and VKORC1-like-1 [VKORC1-L1]) that differ in both enzymatic properties and tissue distribution. For example, Westofen et al. showed that VKORC-L1 has one-third the affinity for 2-3 epoxide vitamin K compared to VKORC1. Subsequent studies have supported the hypothesis that VKOR-L1 is a specialized isoform that protects against oxidative injury through vitamin K regeneration. When cultured HEK293T cells were incubated with H2O2, VKOR-L1 expression increased and evidence of oxidative membrane damage decreased. Clinical observations that calciphylaxis and vitamin K-dependent vascular calcification are more common in the dermis raise the question of whether there is differential expression of VKOR enzymes in the skin. To address this question, Casper et al. determined that mRNA expression of key enzymes is involved in vitamin K regeneration. As shown in Figures 3 and 4, the skin showed the lowest levels of VKOR-C1 compared to any other tissue. Furthermore, NADPH expression in the dermis was below detection level. These observations suggest that any condition or procedure that blocks vitamin K reconstitution (i.e., hemodialysis) makes the tissue more susceptible to pathological calcification.

[0044] The oxidative properties of uremic plasma and the oxidative effects of dialysis itself lead to the "metabolic blockade" and accumulation of 2-3 epoxide vitamin K, as well as a decrease in intracellular levels of vitamin K2. The "downstream" effects of this blockade include the inability of gamma carboxylate key proteins to participate in preventing soft tissue and vascular calcification. The oxidative effects of hemodialysis exacerbate this effect, which may in part explain the tendency of ESRD patients to develop calciphylaxis and vascular calcification.

[0045] Relationship between Vitamin K and Circulating Vitamin K-Dependent Proteins in CKD-ESRD Patients: It is widely recognized that vitamin K levels may not be reduced in ESRD patients despite dietary deficiencies. For example, Holder et al. studied 172 stable dialysis patients and found that only 6% of patients showed clinically significant vitamin K deficiency. However, when the patients' levels of carboxylated osteocalcin were tested, up to 60% had reduced levels. To confirm that this is a common effect of reduced vitamin K activity, the authors also measured another vitamin K-dependent protein, PIVKA-II. Indeed, it was found that up to 90% of both CKD and ESRD patients had reduced levels of carboxylated prothrombin. In a similar study, Pilkey et al. measured vitamin K1 levels in 142 ESRD patients and found that while the majority of patients had adequate vitamin K reserves, 93% had uncarboxylated osteocalcin levels exceeding 20% ​​of the total level. It should be noted that there was no correlation between the total amount of vitamin K1 and the circulating levels of uncarboxylated osteocalcin. This unexpected finding is consistent with the hypothesis that in uric patients, total vitamin K levels may be normal, but the production of reduced vitamin K is blocked by the oxidative properties of uremia.

[0046] In one variant, supplementation or administration of the disclosed composition reverses the hemodialysis-induced inhibition of vitamin K-dependent proteins by normalizing the functionally reduced form of vitamin K. The observation that oxidative conditions can disrupt the vitamin K cycle suggests that the oxidative load occurring during hemodialysis may contribute to the higher rates of vascular and soft tissue calcification observed in the ESRD population. Studies by Himmelfarb et al. and others have confirmed that the simple delivery of hemodialysis can lead to oxidation of several tissue proteins. For example, hydroxyl amino acid side chains are oxidized to carbonyl groups. In studies of CKD and ESRD patients, Himmelfarb et al. demonstrated the use of carbonyl side chain oxidation as a measure of overall oxidative load, and they showed that both CKD and ESRD patients exhibited a significantly higher proportion (15-fold) of carbonyl proteins compared to normal controls (see Figure 5). The proportion of carbonyl proteins is even higher among dialysis patients, which suggests that dialysis not only reduces oxidative load but also contributes to it. As shown in Figure 5, patients with uremia were found to have up to 15 times higher levels of carbonyl proteins. Therefore, the oxidative load generated by hemodialysis delivery leads to the oxidation of functional vitamin K hydroquinone (KH2) to the non-functional natural vitamin. Oxidation of KH2 by hemodialysis blocks its ability to function as a cofactor of GGCX, which downstream leads to a decrease in gamma carboxylation of vitamin K-dependent proteins.

[0047] To confirm that uremia and hemodialysis disrupt the vitamin K cycle, the ratio of vitamin K quinone to 2-3 epoxide vitamin K and vitamin K hydroxyquinone (KH2) can be determined in patients with normal renal function, CKD patients (stages IV and V), and ESRD patients. To determine whether the hemodialysis process further disrupts the vitamin K cycle, the inventors can measure the level of oxidized vitamin K immediately before hemodialysis, then during dialysis (2 hours), and 30 minutes after dialysis. Previous studies investigating the interaction between warfarin and vitamin K metabolism have shown that 2-3 epoxide vitamin K is readily measurable. Patients with CKD and ESRD have higher levels of 2-3 epoxide vitamin K and lower levels of vitamin hydroquinone (KH2) compared to controls. To determine whether the loss of reduced vitamin K (KH2) leads to a decrease in the carboxylation of vitamin K-dependent proteins, the inventors can measure the levels of the following biomarkers in controls, CKD (stages IV and V), and ESRD (pre- and post-hemodialysis): matrix GLA-1 protein; growth arrest-specific gene 6 (Gas-6) protein; PIVAK-II protein; osteocalcin; protein C; protein S; fetuin A; and osteoprotegerin (dialysis plasma level: 6.7 ± 2.2 picomoles / L). The inventors expand these studies by including patients receiving stable 3X / week hemodialysis. Levels of carboxylated and uncarboxylated vitamin K-dependent proteins in pre-dialysis serum can be compared to levels obtained at 2 hours and at the end of the dialysis session. The oxidative effect of dialysis itself leads to a decrease in the levels of carboxylated vitamin K-dependent proteins.

[0048] In one variant, supplementation with the disclosed composition in ESRD patients with uremic arteriolar calcification (calciphylaxis) shortens wound healing time by inhibiting new vascular calcification and restoring blood flow: Skin biopsy: To confirm that supplementation with the disclosed composition prevents the development of small vessel calcification and skin ischemia, the inventors can identify patients with calciphylaxis confirmed by dermal biopsy and randomize patients to treatment with menaquinone-9 or placebo. Clinical endpoints may include: 1) time to release of the wound from vacuum treatment, and 2) wound healing time, defined as the time required for a 50% reduction in the sum of the surface areas of all calciphylaxis wounds.

[0049] Histopathological endpoint: Comparison of diagnostic skin biopsies after 12 weeks of menaquinone-9 treatment with skin biopsies at protocol repeats. Changes in the level of interstitial calcium deposition were defined as changes in von Kossa staining, which can be quantified by digital image color analysis. Skin biopsies can be used to validate biomarkers at the tissue level. This allows for confirmation of the prophylactic properties of MK-9 against early vascular calcification. Validation of these biomarkers in tissue also allows clinicians to utilize biomarkers as a means of tracking clinical response. Microvascular calcification precedes the development of CUA lesions. The level of calcification is quantified by von Kossa calcium staining in peripheral tissues and normalized as calcium content per unit area. The inventors may use von Kossa as a means of confirming the prophylactic properties of MK-9 against the development of vascular calcification.

[0050] In one modified form, supplementation with the disclosed composition reduces wound healing time in ESRD patients with uremic arteriolar calcification (calciphylaxis) by normalizing carboxyprotein C levels in the dermis and preventing primary thrombosis of dermal vessels. Therefore, in one modified form, supplementation or administration of the disclosed composition in diabetic patients prevents the development of vascular dementia by preventing the onset of calcification and microvascular complications.

[0051] Treatment of Alzheimer's disease, apoptosis, and cancer: Alzheimer's disease (AD) is a serious neurodegenerative disorder. Its sporadic forms affect the elderly population (the incidence increases significantly in those aged >75 years), and there are also various familial forms that involve the onset of the disease in the 40s or 50s. AD is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brain of patients. The core component of senile plaques is a small 4kDa amyloid peptide, which is produced by the proteolytic processing of amyloid precursor protein (APP), a large transmembrane protein. Cleavage of APP by beta-secretase (BACE-1) releases a soluble APP-beta fragment, while the 99-amino acid C-terminus remains tethered to the membrane. This C-terminal fragment subsequently undergoes proteolytic processing by gamma-secretase (a membrane polyenzyme complex) to produce amyloid peptides of varying lengths, mainly 40 and 42 amino acids long (Hardy J. et al. (2002) Science; 297 (5580):353-356). In one embodiment, the treatment of diseases or conditions such as Alzheimer's disease, mild cognitive impairment, impaired glucose tolerance, or type 2 diabetes using the composition disclosed herein is mediated by BACE-1, BACE-2, or cathepsin D activity. In one variant, the method disclosed herein may be used to treat calcification in the brain.

[0052] Beta-amyloid (Aβ) has been shown to cause neuronal death by promoting apoptosis and also by direct toxicity. (Hadipour, E. et al. Vitamin K2 Protects PC12 Cells against Aβ (1-42) and H2O2-Induced Apoptosis via P38 MAP Kinase Pathway. Nutr. Neurosci. 2020, 23, 343-352.) Neurotoxicity occurs through various mechanisms, including disruption of calcium homeostasis, oxidative stress, and mitochondrial dysfunction. In PC12 cells derived from rat pheochromocytoma, vitamin K2 prevented neuronal death caused by Aβ(1-42), the most neurotoxic form of Aβ. Experiments also demonstrated that when cells were exposed to either hydrogen peroxide (H2O2) or Aβ(1-42), cells pre-treated with vitamin K2 showed less pronounced apoptosis. Pre-treatment with vitamin K2 also reduced the levels of apoptosis signaling proteins, including a lower Bax / Bcl-2 ratio, decreased the presence of reactive oxygen species (ROS), and increased the levels of the antioxidant glutathione. (Yagami, T. Gas6 Rescues Cortical Neurons from Amyloid β Protein-Induced Apoptosis Neuropharmacology 2002, 43, 1289-1296). The researchers determined that inactivation of the p38MAP kinase pathway is a mechanism related to the potential prophylactic role of VK2 in Alzheimer's disease (AD).

[0053] Researchers observed that increasing the concentration of vitamin K2 (VK2) extended cell survival, presumably because it protected cells from Aβ-induced neuronal death. This effect was reversible upon the addition of warfarin, which was also found to inhibit vitamin K-dependent carboxylation. VK2 was observed to reduce the number of ROS in a dose-dependent manner, and at a concentration of 10 mcmol / L, and reduced the activity of caspase-3, an enzyme that mediates Aβ-induced apoptosis, by 2.5 times. The authors also found that Gas6 plays a role in VK2 protection from Aβ cytotoxicity, which was supported by measured Ca(2+) influx, chromatin condensation, and DNA fragmentation as markers for Aβ neurotoxicity and apoptosis in rat embryonic neuronal cell cultures. Gas6 inhibited Ca(2+) influx in a dose-dependent manner and significantly reduced the amount of Aβ-induced chromatin condensation and DNA fragmentation. Therefore, a clear correlation exists between the antioxidant and anti-apoptotic properties of vitamin K2, which is related to the process of programmed cell death as an effective mechanism in cancer treatment.

[0054] Targeting apoptosis is also effective against various types of cancer, as apoptosis evasion is a prominent characteristic of cancer. Apoptosis is also non-specific to the cause or type of cancer. Villa-Pulgarin JA et al. Mitochondria and lipid raft-located F oSee also F1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine. PLoS Negl. Trop. Dis. 2017;11:e0005805. doi: 10.1371 / journal.pntd.0005805; and Elmore S. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 2007; 35:495-516. doi: 10.1080 / 01926230701320337.

[0055] Vitamin K2 has also been used clinically to complement cancer treatment. (XV, F. et al., Research Progress on the anticancer effects of vitamin K2 (Review). Oncol. Lett. 2018, 15, 8926-8934.) Furthermore, vitamin K2 supplementation has been found to inhibit the growth and metastasis of several cancer lines.See Xia, J. et al., The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells. J. Nutr. Biochem. 2012, 23, 1668-1675; Showalter, S.L. et al., Naturally occurring K vitamins inhibit pancreatic cancer cell survival through a caspase-dependent pathway. J. Gastroenterol. Hepatol. 2010, 25, 738-744. Enomoto, M. et al. Vitamin K2-induced cell growth inhibition via autophagy formation in cholangiocellular carcinoma cell lines. Int. J. Mol. Med. 2007, 20, 801-808; Jinghe, X., Vitamin K and hepatocellular carcinoma: The basic and clinic. World J. Clin. Cases 2015, 3, 757-764. Sada, E. et al. Vitamin K2 modulates differentiation and apoptosis of both myeloid and erythroid lineages. Eur. J. Haematol. 2010, 85, 538-548; Yaguchi, M. et al., Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid. Leukemia 1997, 11, 779-787.Therefore, in one variant, a method is provided for treating cancers such as prostate cancer by administering a therapeutically effective amount of the disclosed composition to control or reduce high levels of hypocarboxylation of osteocalcin.

[0056] In fact, it was determined that intake of MK-4 and MK5-MK-9 was inversely correlated with cancer mortality. In particular, in a cohort study of men, there was an inverse correlation between the incidence of advanced prostate cancer and intake of MK, especially MK-5-MK-9. Nimptsch, K. et al. (2008) Dietary intake of vitamin K and risk of prostate cancer in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg). Am. J. Clin. Nutr. 87, 985-992. Nimptsch, K. et al. (2010) Dietary vitamin K intake in relation to cancer incidence and mortality: results from the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg). Am. J. Clin. Nutr. 91, 1348-1358. In a nested case-control follow-up study, hypocarboxylation of osteocalcin (a marker of inadequate vitamin K status) was significantly higher in advanced or high-grade prostate cancer compared to controls. Nimptsch, K. et al. (2009) Serum undercarboxylated osteocalcin as biomarker of vitamin K intake and risk of prostate cancer: a nested case-control study in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition. Cancer Epidemiol. Biomark. Prev. 18, 49-56.Wang et al. also reported a positive correlation between MK intake and the risk of breast cancer incidence and death, particularly in cases of tubular triple-negative breast cancer and early-stage breast cancer. Wang, K. et al. (2021) Vitamin K intake and breast cancer incidence and death: results from a prospective cohort study. Clin. Nutr. 40, 3370-3378.

[0057] The pharmaceutical compositions disclosed herein may be formulated as liquids or lyophilized powders for parenteral administration. Powders may be reconstituted before use by the addition of suitable diluents or other pharmaceutically acceptable carriers. Liquid formulations are generally buffered isotonic aqueous solutions. Examples of suitable diluents include ordinary isotonic saline solutions, 5% dextrose in water, or buffered sodium or ammonium acetate solutions. Such formulations are particularly suitable for parenteral administration but can also be used for oral administration. Additives such as polyvinylpyrrolidinone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride, or sodium citrate may also be added. Alternatively, these compositions may be encapsulated for oral administration, tableted, or prepared as emulsions or syrups. The compositions may be enhanced or stabilized by the addition of pharmaceutically acceptable solid or liquid carriers, or the preparation of the compositions may be facilitated. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, or water. Solid carriers include starch, lactose, calcium sulfate dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, acacia, agar, or gelatin. The carrier may also contain sustained-release substances such as glyceryl monostearate or glyceryl distearate, either alone or with wax. The amount of solid carrier varies, but can range from approximately 20 mg to approximately 1 g per dose unit. Pharmaceutical preparations are prepared according to conventional pharmacopoeia techniques, including milling, mixing, granulation, and compression as necessary to form tablets, or milling, mixing, and filling to form hard gelatin capsules. When liquid carriers are used, preparations are in the form of syrups, elixirs, emulsions, or aqueous or non-aqueous suspensions. Such liquid formulations may be administered directly by PO or filled into soft gelatin capsules.Suitable formulations for each of these methods of administration can be found, for example, in Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0058] As disclosed herein, the disclosed compositions include oleic acid, Kolliphor® EL (polyoxyl castor oil or Cremophor EL), vitamin E TPGS (D-α-tocopherol polyethylene glycol-1000 succinate), Maisine® CC (glyceryl monolinoleate), Gelucire® 44 / 14 (lauroyl polyoxyl-32 glyceride), Miglyol® 812N (ester of caprylic fatty acid derived from saturated coconut and palm kernel oil with glycerin), Plurol® Oleique (polyglyceryl-6 dioleate), Lauroglycol® 90 (propylene glycol monolaurate (type II)), Labrasol® (caprylocaproyl propyl phosphate) It may also contain solubility enhancers or solubilizers selected from medium-chain monoglycerides and diglycerides, such as polyoxyl-8 glyceride, Kolliphor® EL (polyoxyl castor oil), Captisol® (SBE-beta-cyclodextrin), Encapsin® HPB (hydroxypropyl beta-cyclodextrin), Peceol® (glyceryl monooleate / glyceryl (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil® M2125CS (linoleyl polyoxyl-6 glyceride), and medium-chain monoglycerides and diglycerides.

[0059] In addition to the exemplary embodiments, aspects, and variations described above, further embodiments, aspects, and variations will become apparent by referring to the drawings and figures (FIG) and by examining the following description further. [Modes for carrying out the invention]

[0060] Detailed description of the invention Definition: Unless otherwise specifically stated herein, the definitions of terms used are standard definitions used in the fields of organic synthesis and pharmaceutical science. Exemplary embodiments, aspects, and variations are illustrated in the figures and drawings, and the embodiments, aspects, and variations, as well as the figures and drawings disclosed herein, are intended to be considered illustrative and not limiting.

[0061] As used herein, the term “composition” is used interchangeably with “formulation” and refers to the nanoparticles described herein.

[0062] As used herein, the term "nanoparticle" refers to a particle having at least one dimension less than 1,000 nm, as determined, for example, by a dynamic light scattering particle size analyzer. Other known methods in the art include disk centrifugation, nanoparticle tracking analysis, adjustable resistance pulse sensing, or electron microscopy.

[0063] Generally as used herein, "poorly soluble drugs" means drugs having a solubility of 100 mg / mL or less in water. Another definition may include drugs having a solubility of less than 1 mg / mL across the physiological pH range.

[0064] A “stable” formulation or composition is one in which vitamin K2 essentially retains at least one of its physical stability, chemical stability, and biological activity for at least 6 months or at least 12 months when stored at room temperature. Various analytical techniques for measuring the stability of drug particles are available in the art, such as those specified in Jones, A. et al. Adv. Drug Delivery Rev., 10: 29-90 (1993). In one embodiment, stability can be measured for a selected period and at a selected temperature.

[0065] As used herein, the term "suspension" generally refers to a dispersion of fine particles in a liquid.

[0066] As used herein, the term "emulsion" generally refers to a mixture of two normally immiscible liquids, one of which is colloidally suspended in the other (defining the dispersed phase). The particle size of the dispersed phase in an emulsion is generally between several hundred nanometers and several tens of micrometers.

[0067] "Pharmacologically acceptable salts" means salt compositions that are generally considered to have the desired pharmacological activity, are considered safe and non-toxic, and are acceptable for veterinary and human pharmaceutical applications. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, or with organic acids such as acetic acid, propionic acid, hexanoic acid, malonic acid, succinic acid, malic acid, citric acid, gluconic acid, and salicylic acid.

[0068] "Therapeutic dose" means the amount of compound or composition that exerts any of the biological effects listed herein. [Brief explanation of the drawing]

[0069] [Figure 1] Figure 1 shows the chromatograms of menaquinone-7 and its positional isomers, displayed in a 3:1 ratio, as determined by 1H NMR.

[0070] [Figure 2] Figure 2 is a scheme illustrating the oxidation of KH2-functional carboxylation of vitamin K-dependent proteins induced by uremia and dialysis.

[0071] [Figure 3] Figure 3 is a graph showing VKORC1 in arbitrary units and specific tissues.

[0072] [Figure 4] Figure 4 is a graph showing NADPH levels in arbitrary units and in specific tissues.

[0073] [Figure 5] Figure 5 is a graph showing that patients with CKD and ESRD have a higher percentage of carbonyl protein compared to normal controls. [Examples]

[0074] experiment: 1. Dissolution test of EPN-901 (MK-9) nano-suspended material in simulated feeding intestinal fluid (FeSSIF). material ·MK-9 - Lot A081 ·MK-9 Nano Suspension - F-10 Lot 1-10 Sodium deoxycholate - SAFC lot SLBT2846 • Deionized water - Barnstead E-Pure filtration system • Zirconium milling beads, 0.4mm - Netzsch lot #1310508 • Prepared with FeSSIF, pH 5 - LPI • Polypropylene tube, 1.5 mL, with screw-on cap; supplied by General Lab. Device • Bead-impact homogenizer (bench scale) • Vortex mixer (bench scale) ·Chemical balance HPLC with a variable wavelength UV detector - Agilent 1100 series • Dynamic light scattering particle size analyzer - Malvern Zetasizer ·Melting equipment - Wankel7000

[0075] MK-9 (menaquinone-9, API) was suspended in an aqueous vehicle containing sodium deoxycholate as a wetting agent. The API concentration was set to 4%, and the wetting agent concentration to 0.4% w / w. The API particles were milled to a particle size of 155 nm using a mixture of zirconium milling beads and a high-shear bead impactor. The solubility of the nano-suspended material was tested in a pH 5 simulated intestinal fluid (FeSSIF) at a concentration of 0.01 mg / mL in a triple-strand system. These results were compared with the solubility of API "as is" (commercially available MK-9) tested under the same conditions. [Table 1]

[0076] procedure Part 1. Nano-Suspension Preparation Process

[0077] 1. Set up the equipment and materials indoors by irradiating them with low-wavelength yellow light.

[0078] 2. Add 0.040 g of MK-9 and 0.005 g of sodium deoxycholate to a 1.5 mL polypropylene tube with a screw-on cap.

[0079] 3. Dilute with deionized water to make 1g.

[0080] 4. Vortex for approximately 1 minute to completely dissolve the sodium deoxycholate and uniformly suspend the MK-9.

[0081] 5. Add 0.4 g of 0.4 mm zirconium beads to this suspension.

[0082] 6. Cover the suspension tube with aluminum foil to protect the MK-9 from light.

[0083] 7. Place this tube in an ice bath.

[0084] 8. Set up the bead shocker mixer in a freezer at -20°C.

[0085] 9. Mix the suspension in a freezer at -20°C using a bead impactor at 12 intervals of 5 minutes each. Monitor the temperature of the suspension using a laser thermometer and maintain the suspension at a temperature below 30°C.

[0086] 10. Particle size is measured by dynamic light scattering by diluting 10 μL of the suspension in 950 μL of 0.5% sodium deoxycholate vehicle in a clear cuvette.

[0087] 11. Confirm that the particle size is less than 300 nm.

[0088] 12. Using amber glassware, analyze the assay and impurities in a triple-band RP-HPLC system.

[0089] 14. Store the nano-suspended material at 2-8°C. [Table 2-1] [Table 2-2] result [Table 3-1] Figure 6. Example of a chromatogram of an F-10 nano-suspended material (MK-7 was used as a representative sample). The total amount of impurities detected is less than 0.05% of the total peak area. [ka] [Table 3-2]

[0090] Part 2. In vitro release test

[0091] 1. Clean the dissolution system with DI water.

[0092] Preparation of FeSSIF:

[0093] 2. Fill three containers with 500 mL of FeSSIF medium and heat to 37°C.

[0094] 3. Set the following dissolution conditions:

[0095] [Table 4]

[0096] 4. Vortex the suspension x for approximately 1 minute to make the suspension uniform. Weigh three aliquots of 0.15 g of the 4% nano-suspension into a tare-weighed syringe to obtain the target 100% dissolution concentration of 0.01 mg / mL. Record the weight.

[0097] 5. Add the weighed suspension directly to each container.

[0098] 6. Start the timer.

[0099] After 7.5 minutes, manually withdraw 3 mL from each dissolution container using a syringe.

[0100] Attach the 8.0.2 μm filter to the syringe.

[0101] Transfer 9.2 mL of the dissolved sample to the container and return it to the container.

[0102] 10. Transfer the last 1 mL of sample to the amber HPLC vial and crimp the seal.

[0103] 11. Repeat steps 8-11 at 10, 15, 30, 45, and 60 minutes.

[0104] 12. After 60 minutes, mix each container in a high-shear mixer for 5 minutes.

[0105] Repeat steps 13.8 to 11 to label this sample as "infinite".

[0106] 14. Using a replicated HPLC standard solution, the sample is subjected to HPLC for 10 minutes.

[0107] result [Table 5]

[0108] The % dissolution profiles of MK-9 API and nanosuspendes in FeSSIF show that the F-10 nanosuspend of MK-9 is approximately 98% after approximately 3 minutes and remains approximately 98% after approximately 120 minutes, while MK-9 API shows approximately 20% dissolution after approximately 10 minutes and remains substantially at approximately 20% after 120 minutes.

[0109] Nanomilled particles dissolve at significantly faster rates and in greater quantities than "as-is" API particles or MK-9 API.

[0110] 2. Summary of the dissolution test of MK-9 nanosuspends in simulated feeding intestinal fluid (FeSSIF).

[0111] MK-9 (menaquinone-9, API) is suspended in an aqueous vehicle containing sodium deoxycholate as a wetting agent. The API concentration is 4%, and the wetting agent concentration is 0.4% w / w. The API particles are milled to a particle size of 155 nm using a mixture of zirconium milling beads and a high-shear bead impactor. The dissolution of the nano-suspending is tested in FeSSIF at pH 5 at a concentration of 0.01 mg / mL in a triple chamber. These results are compared to the dissolution of "as-is" API tested under the same conditions. The nano-milled particles dissolve at a significantly faster rate and in a larger volume than the "as-is" API particles. The nano-milling process is scaled up, the suspended nanoparticles are removed from the suspension vehicle, dried, formulated into rapid-dissolving tablets, and tested.

[0112] Administration of nanoparticle compositions to subjects at risk of developing calciphylaxis: This embodiment describes the administration of the compositions of this application to subjects at risk of developing calciphylaxis but who have not yet developed the characteristic skin lesions of calciphylaxis. Possible risk factors include, but are not limited to, diabetes, obesity, hemodialysis, and prior treatment with warfarin (Nigwekar et al. (2016) A Nationally Representative Study of Calcific Uremic Arteriolopathy Risk Factors, J. AM. SOC. NEPHROL. 27(11):3421-9). Administration of these compositions may result in protection of subjects from skin lesions and changes in certain biomarker levels that serve as indicators of prevention of calciphylaxis development.

[0113] Subjects at risk of developing calciphylaxis are orally administered 0.1 mg, 3 mg, 5 mg, or 10 mg of the selected composition of this application once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is 0.1 mg, 3 mg, 5 mg, or 10 mg in pill or soft gel capsule form. Two 25 mg capsules would be administered once daily to a 50 mg dose cohort. It should be noted that not all subjects with high risk factors for calciphylaxis will develop the characteristic skin lesions of calciphylaxis. The intention of proactive treatment with the composition of this application (before clinical diagnosis of calciphylaxis) is the prevention of lesion appearance. Therefore, a reduction or elimination of the frequency of lesion appearance is intended to be the measure of success.

[0114] Several biomarkers can be evaluated to determine the potency of the composition administered at three different dose levels. Exemplary biomarkers include PIVKA-II; uncarboxylated matrix Gla protein and total matrix Gla protein (MGP); uncarboxylated osteocalcin protein, carboxylated osteocalcin protein and total osteocalcin protein; uncarboxylated protein C, carboxylated protein C and total protein C; osteoprotegerin, fetuin A and hs-CRP. Blood samples are obtained and biomarkers are measured according to the following schedule. Blood sampling can be performed between weekly or monthly treatments. Administration of the disclosed composition results in (i) a decrease in PIVKA-II, an indicator of slowing, stopping, or reversing the progression of calciphylaxis; and (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin, and / or uncarboxylated protein C, also indicators of slowing, stopping, or reversing the progression of calciphylaxis. Furthermore, pulse wave velocity (PWV) can be measured to assess arterial compliance. Improved vascular compliance is an indicator of slowing, stopping, or reversing the progression of calciphylaxis.

[0115] Administration of the disclosed compositions of this application to subjects diagnosed with calciphylaxis: This example describes the administration of the disclosed composition to a subject diagnosed with calciphylaxis. Typical symptoms include the presentation of characteristic painful skin lesions (Nigwekar et al. (2015) Calciphylaxis: Risk Factors, Diagnosis, and Treatment. Am. J. Kidney Dis. 66:133-46). A definitive diagnosis of calciphylaxis is made by skin biopsy.

[0116] Subjects diagnosed with calciphylaxis receive orally once daily 0.1 mg, 3 mg, 5 mg, or 10 mg of the disclosed composition for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is 0.1 mg, 3 mg, 5 mg, or 10 mg tablets or soft gel capsules. Two 25 mg capsules are administered once daily to a 50 mg dose cohort.

[0117] The cessation or reduction of lesion size and frequency is intended to be an indicator of successful treatment. Administration of the disclosed composition as described above results in the cessation or reduction of lesion size and frequency. Furthermore, since calciphylaxis carries a significant risk of death, an increase in overall survival of diagnosed subjects would be an indicator of treatment success. Furthermore, administration of the disclosed composition as described above results in an increase in overall survival of diagnosed subjects.

[0118] Administration of the disclosed composition to subjects with end-stage renal disease (ESRD) to reverse or slow the progression of tissue calcification: This embodiment describes the administration of the disclosed composition to subjects with ESRD undergoing stable hemodialysis. Administration of the disclosed composition results in changes in certain biomarker levels that indicate aortic compliance (as measured by plethysmography), vascular calcification, and slowing, cessation, or reversal of the progression of tissue calcification.

[0119] Patients with ESRD receiving stable hemodialysis receive 0.1 mg, 3 mg, 5 mg, or 10 mg of the disclosed composition orally once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is 0.1 mg, 3 mg, 5 mg, or 10 mg tablets or soft gel capsules. Two 25 mg capsules are administered once daily to a 50 mg dose cohort.

[0120] A 50-year-old, 65kg male patient diagnosed with typical symptoms associated with moderate calciphylaxis is treated with a 0.1mg composition for 8 weeks. After the treatment period, the patient is hospitalized for evaluation. The patient is found to have significant changes in the levels of the biomarkers tested, suggesting an approximately 10% reduction in vascular calcification and also showing a 10% reduction in tissue calcification.

[0121] A 65-year-old, 45kg female patient diagnosed with typical symptoms associated with moderate calciphylaxis is treated with a 0.1mg composition for 10 weeks. After the treatment period, the patient is hospitalized for evaluation. The patient is found to have significant changes in the levels of the biomarkers tested, suggesting an approximately 20% reduction in vascular calcification and also showing a 15% reduction in tissue calcification.

[0122] A 55-year-old, 70kg male patient diagnosed with typical symptoms associated with moderate calciphylaxis was treated with a 0.2mg composition for 3 months. After the treatment period, the patient was hospitalized for evaluation. The patient was found to have significant changes in the levels of the biomarkers tested, suggesting an approximately 25% reduction in vascular calcification and also showing a 20% reduction in tissue calcification.

[0123] The coronary artery calcium score (CAC) is used to estimate the degree of calcification in the thoracic arteries. A high CAC score is an indicator of calcification, and treatment aims to stop, reverse, or slow the rate of increase in the CAC score over a long period.

[0124] Aortic plethysmography is also used to measure arterial compliance, which decreases as calcification increases. Pulse wave velocity (PWV) is also measured to assess arterial compliance. The above measures are useful in estimating the effectiveness of treatments intended to prevent, slow, stop, or reverse vascular calcification. These measurements are used before and after treatment with the disclosed composition to evaluate the value of the treatment.

[0125] Furthermore, several biomarkers are evaluated to determine the potency of the disclosed compositions at three dose levels. Exemplary biomarkers include PIVKA-II; uncarboxylated matrix Gla protein and total matrix Gla protein (MGP); uncarboxylated osteocalcin protein, carboxylated osteocalcin protein and total osteocalcin protein; uncarboxylated protein C, carboxylated protein C and total protein C and hs-CRP. Most conveniently, blood samples are obtained and biomarkers are measured while the patient is at the hospital for hemodialysis.

[0126] Administration of the disclosed composition may result in (i) a decrease in PIVKA-II, which is an indicator of slowing, stopping, or reversing the progression of tissue calcification; (ii) a decrease in non-carboxylated MGP, non-carboxylated osteocalcin, and / or non-carboxylated protein C, which are indicators of slowing, stopping, or reversing the progression of tissue calcification; and / or (iii) a decrease in hs-CRP, which is an indicator of slowing, stopping, or reversing the progression of tissue calcification; and / or a reduction in inflammation. After daily administration of 0.01 mg, 0.1 mg, 3 mg, 5 mg, or 10 mg of the disclosed composition, at least one of PIVKA-II, non-carboxylated matrix Gla protein (MGP), and non-carboxylated osteocalcin protein shows changes that are indicators of slowing, stopping, or reversing the progression of tissue calcification.

[0127] While several exemplary embodiments, aspects, and variations are presented herein, those skilled in the art will recognize certain modifications, alterations, additions, and combinations, as well as certain partial combinations of embodiments, aspects, and variations. The following claims are construed to include all such modifications, alterations, additions, and combinations, and certain partial combinations of embodiments, aspects, and variations are intended to fall within their scope.

Claims

1. A composition comprising water-soluble vitamin K2 nanoparticles, wherein the nanoparticles have an average particle size of about 0.1 nm to 1,000 nm.

2. The composition according to claim 1, wherein the vitamin K2 is MK-9 (menaquinone-9) or menaquinol-9 (MKH2-9).

3. The composition according to claim 2, wherein the vitamin K2 is MK-9.

4. The composition according to any one of claims 1 to 3, wherein the nanoparticles have an average particle size of 200 nm, 175 nm, 150 nm, 125 nm, 115 nm, 100 nm, 90 nm, less than 80 nm, or less than 75 nm.

5. The composition according to any one of claims 1 to 4, wherein the nanoparticles are prepared using a homogenizer selected from the group consisting of a rotor stator homogenizer, a bead mill homogenizer, or a mortar and pestle homogenizer.

6. Poloxamer 188, Polysorbate 80, Polysorbate 20, Vit E-TPGS (TPGS), TPGS-1000, TPGS-750-M, Solutol HS15, PEG-40 Hydrogenated Castor Oil, PEG-35 Castor Oil, PEG-8 Caprylic / Capric Glyceryl, PEG-32 Glyceryl Laurate, PEG-32 Glyceryl Palmitostearate, Polysorbate 85, Polyglyceryl-6 Dioleate, Sorbitan Monooleate, Capmul The composition according to any one of claims 1 to 5, further comprising MCM, Maisine 35-1, glyceryl monooleate, glyceryl monolinoleate, PEG-6 oleate glyceryl, PEG-6 linoleate glyceryl, oleic acid, linoleic acid, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, polyglyceryl-3 diisostearate, and lecithin, wherein the composition further comprises at least one emulsifier selected from the group consisting of MCM, Maisine 35-1, glyceryl monooleate, glyceryl monolinoleate, PEG-6 linoleate, oleic acid, linoleic acid, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, and lecithin.

7. The composition according to claim 6, wherein the emulsifier is selected from polysorbate 80, Vit E-TPGS, Solutol HS15, PEG-40 hydrogenated castor oil, and PEG-35 castor oil.

8. The composition according to any one of claims 1 to 7, further comprising at least one bioavailability activator selected from the group consisting of medium-chain fatty acids, omega-3 fatty acids, capric acid, caprylic acid, alkyl glycosides, chitosan, trimethylated chitosan, ethylene glycol tetraacetic acid, ethylenediamine tetraacetic acid, salicylic acid, genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one)) and pharmaceutically acceptable salts thereof.

9. The composition according to any one of claims 1 to 8, which is a nano-suspension in water.

10. The composition according to any one of claims 1 to 9, wherein the nano-suspended material is at least five times more soluble than commercially available, unhomogeneized vitamin K2.

11. The composition according to claim 10, wherein the vitamin K2 is MK-9.

12. The composition according to claim 10 or 11, wherein the nano-suspended vitamin K2 is carried out at a concentration of 0.01 mg / mL in water.

13. The composition according to claim 10 or 11, wherein the nano-suspended material is present in an aqueous solution in the simulated feeding intestinal fluid (FeSSIF).

14. The composition according to claim 13, wherein the solubility is determined after 10 minutes in FeSSIF.

15. The composition according to any one of claims 1 to 14, further comprising pharmaceutically acceptable additives, and effective for the treatment of vitamin K-related conditions such as osteoporosis or arteriosclerosis.

16. A method for treating a disease in a mammal selected from the group consisting of neurodegenerative diseases, retinopathy, polyarthritis rheumatica, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infection, pathologies associated with aging of the skin and tissues, pathologies associated with mitochondrial dysfunction, and cachexia associated with nutritional deficiencies, wherein the treatment results in an increase in the lifespan of the mammal, and the method comprises administering a therapeutically effective amount of the composition according to any one of claims 1 to 15.

17. A method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative disorders, and cardiovascular diseases, the method comprising the step of administering to the mammal a therapeutically effective amount of the composition according to any one of claims 1 to 15.

18. A method for treating or preventing osteoporosis and / or osteopenia, comprising the step of administering to a patient in need of treatment a therapeutically effective amount of the composition according to any one of claims 1 to 15.

19. A method for treating, preventing, slowing, stopping, and / or reversing the progression of calciphylaxis in a mammal requiring treatment, prevention, slowing, stopping, and / or reversing its progression, comprising the step of administering to the mammal a therapeutically effective amount of the composition according to any one of claims 1 to 15 and a pharmaceutically acceptable additive to prevent, slow, stop, or reverse calciphylaxis.

20. The method according to claim 19, wherein the mammal has distal calciphylaxis and / or central calciphylaxis.

21. The method according to claim 19 or 20, wherein the mammal has diabetes, chronic kidney disease, or end-stage renal disease.

22. The method according to any one of claims 19 to 21, wherein the mammal has chronic obstructive pulmonary disease (COPD).

23. The method according to any one of claims 19 to 22, wherein the mammal has a calciphylaxis-related skin lesion.

24. A method for treating, preventing, slowing, stopping, and / or reversing tissue calcification in a prediabetic mammal (or subject) having diabetes mellitus, chronic kidney disease or a combination thereof, and requiring treatment, prevention, slowing, cessation, and / or reversal of its progression, wherein the method comprises the step of administering to the mammal at least 0.01 mg per day of any one composition of the compositions described in any one of claims 1 to 15, the composition of any one of claims 1 to 15 being administered in a pharmaceutical composition.

25. The method according to claim 24, wherein the mammal has a chronic kidney disease.

26. The method according to claim 17, wherein the proliferative disorder is selected from the group consisting of cancer, leukemia, and inflammatory diseases.

27. A method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative disorders, and cardiovascular diseases, the method comprising the step of administering to the mammal a therapeutically effective amount of the composition according to any one of claims 1 to 15.

28. The method according to claim 26, wherein the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, leukemia, neuroblastoma, glioblastoma, cervix, colorectal cancer, pancreas, bladder, kidney, prostate, ovary, and head and neck cancer.

29. A method for treating, preventing, slowing, stopping, and / or reversing the progression of Alzheimer's disease (AD) in a mammal or subject that requires treatment, prevention, slowing, stopping, and / or reversing the progression of the AD, comprising the step of administering to the mammal or subject at least 0.01 mg per day of any one composition of the compositions described in any one of claims 1 to 15 to prevent, slow the progression of, and / or stop, or reverse the Alzheimer's disease.

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